Translational regulation of parathyroid hormone gene expression and RNA: Protein interactions

The aim of this study was to investigate the mechanism by which translation of parathyroid hormone (PTH) mRNA is regulated with regard to the subcellular distribution of PTH mRNA and RNA:protein interactions. Sucrose density ultracentrifugation of RNA from bovine parathyroid cells indicated that there was no evidence for a pool of nonribosomal PTH mRNA, and the extracellular calcium concentration had no effect on polysome size. UV cross‐linking studies revealed two proteins in parathyroid cell cytosol which bound specifically to the 5′‐untranslated region (UTR) of PTH mRNA with molecular masses of 66 and 68 kD while proteins with apparent molecular masses of 48 and 70 kD bound to the 3′‐UTR. In vitro translation assays indicated that parathyroid cell cytosol contains factors that inhibit translation of PTH mRNA. Fractionation of cytosol revealed that this effect was associated with proteins within the molecular mass range 30–90 kD. To determine which sequences in PTH mRNA mediate translational regulation, RNA was synthesized from luciferase gene constructs containing the 5′‐and/or 3′‐UTR of PTH mRNA, and translated in vitro. Addition of parathyroid cell cytosol reduced the translation of RNA containing the 5′‐ and 3′‐UTR of PTH mRNA by 44 + 7% but had no effect on the translation of RNA containing only the luciferase coding region. Translation of RNA containing only the 5′‐UTR of PTH mRNA was unchanged; however, cytosol reduced the translation of RNA containing the 3′‐UTR by 31 + 9%. These data demonstrate a role for RNA:protein interactions in the regulation of PTH synthesis and that translational control is mediated primarily through interactions with the 3′‐UTR of PTH mRNA.